Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen-deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in plasmon-enhanced fluorescence detection of weak emitting species, and refractive index based single-molecule detection on optoplasmonic sensors. The role of the local field in providing plasmonic signal enhancements for single-molecule detection has been studied in great detail. Nevertheless, few studies have compared the experimental results in both techniques for single-molecule studies. Here we developed the first optical setup that integrates optoplasmonic and DNA-PAINT based detection of oligonucleotides to compare these sub-platforms and provide complementary insights into single molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.

AbstractBackground-free detection of inherently weak chiroptical signals remains one of the great challenges in research communities and industries. We demonstrate coherent multipolar amplification of chiroptical responses via a magnetoelectric nanoparticle capped with an optically active monolayer encapsulated in a lossless background medium. Such an achiral nanoparticle can simultaneously support both electric and magnetic Mie-type resonances. We show how the combined excitation of orthogonal multipolar modes of the same order boosts the magnetoelectric coupling induced by the adsorbed chiral molecules, thus enabling coherently enhanced chiroptical responses from the ligand-capped magnetoelectric nanoparticle and allowing for absolute chirality measurements, in comparison with non-magnetoelectric nanoparticles. Furthermore, we develop rigorous expressions to separate relative contributions of chiral and nonchiral portions of circular differential absorption cross section, and analyzed the chirality-dependent far-field radiation patterns at different overlapped multipolar modes, providing a theoretical framework to understand the underlying enhancement mechanism of the magnetoelectric-assisted sensing of molecular chirality.

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen-deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in plasmon-enhanced fluorescence detection of weak emitting species, and refractive index based single-molecule detection on optoplasmonic sensors. The role of the local field in providing plasmonic signal enhancements for single-molecule detection has been studied in great detail. Nevertheless, few studies have compared the experimental results in both techniques for single-molecule studies. Here we developed the first optical setup that integrates optoplasmonic and DNA-PAINT based detection of oligonucleotides to compare these sub-platforms and provide complementary insights into single molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.

AbstractBackground-free detection of inherently weak chiroptical signals remains one of the great challenges in research communities and industries. We demonstrate coherent multipolar amplification of chiroptical responses via a magnetoelectric nanoparticle capped with an optically active monolayer encapsulated in a lossless background medium. Such an achiral nanoparticle can simultaneously support both electric and magnetic Mie-type resonances. We show how the combined excitation of orthogonal multipolar modes of the same order boosts the magnetoelectric coupling induced by the adsorbed chiral molecules, thus enabling coherently enhanced chiroptical responses from the ligand-capped magnetoelectric nanoparticle and allowing for absolute chirality measurements, in comparison with non-magnetoelectric nanoparticles. Furthermore, we develop rigorous expressions to separate relative contributions of chiral and nonchiral portions of circular differential absorption cross section, and analyzed the chirality-dependent far-field radiation patterns at different overlapped multipolar modes, providing a theoretical framework to understand the underlying enhancement mechanism of the magnetoelectric-assisted sensing of molecular chirality.

Abstract
. Optical atomic clocks with compact size, reduced weight and low power consumption have broad out-of-the-lab applications such as satellite-based geo-positioning and communication engineering. Here, we propose an active optical microclock based on the lattice-trapped atoms evanescently interacting with a whispering-gallery-mode microcavity. Unlike the conventional passive clock scheme, the active operation directly produces the optical frequency standard without the need of extra laser stabilization, substantially simplifying the clock configuration. The numerical simulation illustrates that the microclock’s frequency stability reaches
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. at 1 s of averaging, over one order of magnitude better than the recently demonstrated chip-scale optical clock that is built upon rubidium vapor cell and also more stable than current cesium fountain clocks and hydrogen masers. Our work extends the chip-scale clocks to the active fashion, paving the way towards the on-chip quantum micro-metrology, for example, the optical frequency comparison and synchronization between multiple microclocks through frequency microcombs.

The single-molecule study provides an unprecedented deep view into the biological process, unveiling the hidden heterogeneity that is hard to observe in ensemble methods. Recently, various techniques have shown the detection of biomolecules with single-molecule level sensitivity. However, each technique has its unique advantages and drawbacks. Single-molecule techniques can influence the molecular systems that they intend to detect in different ways. For example, fluorescence labels may affect the kinetics and dynamics of biomolecular interactions, while plasmonic-based approaches use local field enhancements that are not uniform across the involved plasmonic nanostructures, both of which can play a significant role in the observed statistics. Before one can combine the information obtained from fluorescence and optoplasmonic techniques, single-molecule experiments must be compared and cross-validated.

Here we first compare the detection of DNA hybridization on fluorescence-based imaging technique and optoplasmionic sensors. We investigate the impact of (i) the presence of labels, and (ii) the potential influence of the plasmonic nanoparticle surface. Our measurements reveal that the dissociation rates of hybridized DNA strands are approximately the same for both techniques. Our study establishes the equivalence of both techniques for this DNA molecular test system and can serve as the basis for combining these techniques in other single-molecule studies. With optoplasmonic sensing, our results indicate that one may benefit from the larger binding efficiency of fluorescence imaging while the impact of the label is checked with the optoplasmonic sensing platform.


To further combine the two single-molecule characterisation systems, we developed the first optical setup that integrates optoplasmonic and fluorescence-based detection. These sub-platforms provide complementary insights into single-molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient DNA hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.

We hereby present a novel “grafting-to”-like approach for the covalent attachment of plasmonic nanoparticles (PNPs) onto whispering gallery mode (WGM) silica microresonators. Mechanically stable optoplasmonic microresonators were employed for sensing single-particle and single-molecule interactions in real time, allowing for the differentiation between binding and non-binding events. An approximated value of the activation energy for the silanization reaction occurring during the “grafting-to” approach was obtained using the Arrhenius equation; the results agree with available values from both bulk experiments and ab initio calculations. The “grafting-to” method combined with the functionalization of the plasmonic nanoparticle with appropriate receptors, such as single-stranded DNA, provides a robust platform for probing specific single-molecule interactions under biologically relevant conditions.

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic
nanoparticles is key in plamon enhanced fluorescence detection from weak
emitting species, and refractive index based single-molecule detection on
optoplasmonic sensors. The role of the local field in providing plasmonic
signal enhancements for single-molecule detection has been studied in great
detail. Nevertheless, few studies have compared the experimental results in
both techniques for single-molecule studies. Here we developed the first
optical setup that integrates optoplasmonic and DNA-PAINT based detection of
oligonucleotides to compare these sub-platforms and provide complementary
insights into single-molecule processes. We record the fluorescence and
optoplasmonic sensor signals for individual, transient hybridisation events.
The hybridisation events are observed in the same sample cell and over a
prolonged time (i.e. towards high binding site occupancies). A decrease in the
association rate over the measurement duration is reported. Our dual
optoplasmonic sensing and imaging platform offers insight into the observed
phenomenon, revealing that irreversible hybridisation events accumulate over
detected step signals in optoplasmonic sensing. Our results point to novel
physicochemical mechanisms that result in the stabilisation of DNA
hybridisation on optically-excited plasmonic nanoparticles.

Coherent amplification of chiroptical activity from a molecularly-thin optically-active substance has been a long-standing challenge due to the inherently weak nature of chiral responses. Here we report how a coherent perfect absorber (CPA) enabled by an achiral optical system obeying parity-time (PT) symmetry has an enhanced ability to effectively sense molecular chirality of monolayered substances. We demonstrate that such a CPA-based PT-symmetric system enables us in complete darkness to probe a subtle signal change induced by the introduction of a small disturbance, such as adsorbed chiral monolayer, to the unperturbed PT-symmetric system, and allows for absolute measurement and quantitative detection of the magnitude and sign of both real and imaginary parts of the chirality parameter in a background-free environment. Moreover, the CPA-based PT-symmetric system also exhibits three orders of magnitude enhancement in chiroptical responses of molecules, which is consistent with analytical calculations of differential absorption.

Enzymes catalyze most of the biochemical reactions in our cells. The functionality of enzymes depends on their dynamics starting from small bond vibrations in the fs timescale to large domain motions in the microsecond-millisecond timescale. Understanding the precise and rapid positioning of atoms within a catalytic site by an enzyme’s molecular movements is crucial for understanding biomolecular processes and for realizing synthetic biomolecular machines in the longer term. Hence, sensors capable of studying enzymes over a wide range of amplitudes and timescale and ideally one enzyme at a time are required. Many capable single-molecule techniques have been established in the past three decades, each with its pros and cons. This thesis presents the development of one such single-molecule sensor. The sensor is based on plasmonically enhanced whispering gallery mode resonators and is capable of studying enzyme kinetics and large-scale dynamics over the timescale of ns-seconds. Unlike fluorescence techniques which require labeling of the enzymes with dyes, the technique presented in this work detects single enzymes immobilized on the surface of plasmonic gold nanoparticles. A fast, low-noise, lock-in method is utilized to extract sensor signals in the microsecond timescale. Using a model enzyme, the ability of the sensor to detect conformational fluctuations of single enzymes is shown. Further, the thermodynamics of the enzyme is studied and the relevant thermodynamic parameters are extracted from the single-molecule data. Additionally, we extract the heat capacity changes associated with the enzyme using the single-molecule data. The sensor system presented in this thesis in the future could enable a fast, real-time, rapid throughput, lab-on-chip sensor system for studying single enzymes for both research and clinical use.

Abstract
. Quantum-enhanced sensing and metrology pave the way for promising routes to fulfil the present day fundamental and technological demands for integrated chips which surpass the classical functional and measurement limits. The most precise measurements of optical properties such as phase or intensity require quantum optical measurement schemes. These non-classical measurements exploit phenomena such as entanglement and squeezing of optical probe states. They are also subject to lower detection limits as compared to classical photodetection schemes. Biosensing with non-classical light sources of entangled photons or squeezed light holds the key for realizing quantum optical bioscience laboratories which could be integrated on chip. Single-molecule sensing with such non-classical sources of light would be a forerunner to attaining the smallest uncertainty and the highest information per photon number. This demands an integrated non-classical sensing approach which would combine the subtle non-deterministic measurement techniques of quantum optics with the device-level integration capabilities attained through nanophotonics as well as nanoplasmonics. In this back drop, we review the underlining principles in quantum sensing, the quantum optical probes and protocols as well as state-of-the-art building blocks in quantum optical sensing. We further explore the recent developments in quantum photonic/plasmonic sensing and imaging together with the potential of combining them with burgeoning field of coupled cavity integrated optoplasmonic biosensing platforms.

AbstractLasers are the pillars of modern optics and sensing. Microlasers based on whispering-gallery modes (WGMs) are miniature in size and have excellent lasing characteristics suitable for biosensing. WGM lasers have been used for label-free detection of single virus particles, detection of molecular electrostatic changes at biointerfaces, and barcode-type live-cell tagging and tracking. The most recent advances in biosensing with WGM microlasers are described in this review. We cover the basic concepts of WGM resonators, the integration of gain media into various active WGM sensors and devices, and the cutting-edge advances in photonic devices for micro- and nanoprobing of biological samples that can be integrated with WGM lasers.

Chiral molecules are ubiquitous in nature; many important synthetic chemicals and drugs are chiral. Detecting chiral molecules and separating the enantiomers is difficult because their physiochemical properties can be very similar. Here we review the optical approaches that are emerging for detecting and manipulating chiral molecules and chiral nanostructures. Our review focuses on the methods that have used plasmonics to enhance the chiroptical response. We also review the fabrication and assembly of (dynamic) chiral plasmonic nanosystems in this context.

AbstractResearchers in the field of whispering-gallery-mode (WGM) microresonators have proposed biointegrated low-threshold WGM lasers, to enable large-scale parallel single-cell tracking and barcoding. Although the reported devices have so far been primarily investigated in model applications, most recent results represent important steps towards the development of in vivo tags and sensors that utilize the unique and narrow spectral features of miniature WGM lasers.

he term whispering gallery mode (WGM) was first introduced to describe the curvilinear propagation of sound waves under a cathedral dome. The physical concept has now been generalized to include light waves that are continuously reflected along the closed concave surface of an optical cavity such as a glass microsphere. The circular path of the internally reflected light results in constructive interference and optical resonance, a morphology-dependent resonance that is suitable for interferometric sensing. WGM resonators are miniature micro-interferometers that use the multiple-cavity passes of light for very sensitive measurements at the microscale and nanoscale, including single-molecule and ion measurements. This Primer introduces various WGM sensors based on glass microspheres, microtoroids, microcapillaries and silicon microrings. We describe the sensing mechanisms, including mode splitting and resonance shift, exceptional-point-enhanced sensing and optomechanical and optoplasmonic signal transductions. Applications and experimental results cover in vivo and single-molecule sensing, gyroscopes and microcavity quantum electrodynamics. We also discuss data analysis methods and the limitations of WGM techniques. Finally, we provide an outlook for molecule, in vivo and quantum sensing.

Early detection is critical to the successful treatment of life-threatening infections caused by fungal pathogens, as late diagnosis of systemic infection almost always equates with a poor prognosis. The field of fungal diagnostics has some tests that are relatively simple, rapid to perform and are potentially suitable at the point of care. However, there are also more complex high-technology methodologies that offer new opportunities regarding the scale and precision of fungal diagnosis, but may be more limited in their portability and affordability. Future developments in this field are increasingly incorporating new technologies provided by the use of new format biosensors. This overview provides a critical review of current fungal diagnostics and the development of new biophysical technologies that are being applied for selective new sensitive fungal biosensors to augment traditional diagnostic methodologies.

Biomolecules can be detected through induced changes in the optical whispering-gallery mode (WGM) resonance in a circularly symmetric dielectric. The spatial and temporal confinement of light in a WGM is further complemented by coupling to the localised surface plasmons (LSPs) of metallic nanoparticles attached to the WGM resonator. LSP-WGM hybridisation allows for the optical readout of single-molecule surface reactions on gold nanoantennae, the mechanisms for which are not yet fully understood from a theoretical perspective. The specificity of this modality is, moreover, a subject of intense research. In this thesis, we propose three strategies for characterising molecules with light. The first strategy is a prototype polarimeter that differentiates chirality based on a signal-reversible Faraday effect in a magneto-optical WGM microcavity. Thermal tuning integrated into the resonator minimises geometrical birefringence, in turn maximising Faraday rotation to optimise chiral sensitivity. There we endeavour to resolve single-molecule chirality. Without engineering reconsiderations, however, the polarimeter is found to be limited to bulk chiral analysis. The second strategy is an (optoplasmonic) LSP-WGM resonator with chiral gold nanoantennae. Signals from the molecules conjointly show a correlation with the molecular weight and diffusivity of detected DL-cysteine and poly-DL-lysine. Aside from these features, the sensing site heterogeneity on the chiral gold nanoparticles impedes chiral discrimination. The third strategy is a novel reaction scheme adapted to the optoplasmonic sensor. Aminothiol linkers functionalise the gold surface via amine-gold anchoring, setting up cyclical interactions with thiolated analytes by thiol/disulfide exchange. Unexpected perturbations in the LSP-WGM resonance are observed, such as linewidth oscillation without resonance shift attributed to optomechanical coupling between LSPs and the vibrational modes in a given analyte. This offers a new form of spectroscopy wherein single biomolecules could be characterised by their mass, size, and composition through monitoring secondary parameters of the optoplasmonic resonance.

Here, we report shifts of the linewidth of a plasmon enhanced whispering gallery mode (WGM) of a glass microsphere cavity due to binding of single sub-kDa molecules. The observed linewidth of the WGM can either increase or decrease upon binding of single molecules depending on the location of their binding sites. The linewidth shifts arise due to the change in the unresolved frequency splitting of standing wave modes (SWMs). These SWMs are formed due to the scattering from the gold nanoparticles attached to the WGM. Monitoring the WGM linewidth provides a robust method for sensing single molecules over prolonged time periods as the linewidth is unaffected by changes in the host refractive index due to drifts in temperature, pressure, or change in the concentration of buffers.

Tailoring of the band gap in semiconductors is essential for the development of novel devices. In standard semiconductors, this modulation is generally achieved through highly energetic ion implantation. In two-dimensional (2D) materials, the photophysical properties are strongly sensitive to the surrounding dielectric environment presenting novel opportunities through van der Waals heterostructures encompassing atomically thin high-κ dielectrics. Here, we demonstrate a giant tuning of the exciton binding energy of the monolayer WSe2 as a function of the dielectric environment. Upon increasing the average dielectric constant from 2.4 to 15, the exciton binding energy is reduced by as much as 300 meV in ambient conditions. The experimentally determined exciton binding energies are in excellent agreement with the theoretical values predicted from a Mott-Wannier exciton model with parameters derived from first-principles calculations. Finally, we show how texturing of the dielectric environment can be used to realize potential-well arrays for excitons in 2D materials, which is a first step toward exciton metamaterials.

This interdisciplinary book covers the fundamentals of optical whispering gallery mode (WGM) microcavities, light–matter interaction, and biomolecular structure with a focus on applications in biosensing. Novel biosensors based on the hybridization of WGM microcavities and localized surface plasmon resonances (LSPRs) in metal nanoparticles have emerged as the most sensitive microsystem biodetection technology that boasts single molecule detection capability without the need for amplification and labeling of the analyte. The book provides an ample survey of the physical mechanisms of WGMs and LSPRs for detecting affinity, concentration, size, shape and orientation of biomarkers, while informing the reader about different classes of biomolecules, their optical properties and their importance in label-free clinical diagnostics. For the more advanced reader, advanced applications of WGMs and LSPRs in exploring the fundamental nature of quantum physics are discussed.

AbstractProbing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

We report the experimental realization of magneto-optical coupling between whispering-gallery modes in a germanate (56GeO2-31PbO-9Na2O-4Ga2O3) microspherical cavity due to the Faraday effect. An encapsulated gold conductor heats the resonator and tunes the quasi-transverse electric (TE) and quasi-transverse magnetic (TM) polarized modes with an efficiency of ∼ 65 fm/V at a peak-to-peak bias voltage of 4 V. The signal parameters for a number of heating regimes are quantified to confirm sensitivity to the generated magnetic field. The quasi-TE and quasi-TM resonance frequencies stably converge near the device's heating rate limit (equivalently, bias voltage limit) in order to minimize inherent geometrical birefringence. This functionality optimizes Faraday rotation and thus enables the observation of subsequent magneto-optics.

Monitoring the conformational dynamics of proteins is crucial for a better understanding of their biological functions. To observe the structural dynamics of proteins, it is often necessary to study each molecule individually. To this end, single-molecule techniques have been developed such as Förster resonance energy transfer and optical tweezers. However, although powerful, these techniques do have their limitations, for example, limited temporal resolution, or necessity for fluorescent labelling, and they can often only access a limited set of all protein motions. Here, within the context of established structural biology techniques, we review a new class of highly sensitive optical devices based on WGM, which characterise protein dynamics on previously inaccessible timescales, visualise motions throughout a protein, and track movements of single atoms.

The noninvasive monitoring of protein secretion of cells responding to drug treatment is an effective and essential tool in latest drug development and for cytotoxicity assays. In this work, a surface functionalization method is demonstrated for specific detection of protein released from cells and a platform that integrates highly sensitive optical devices, called whispering-gallery mode biosensors, with precise microfluidics control to achieve label-free and real-time detection. Cell biomarker release is measured in real time and with nanomolar sensitivity. The surface functionalization method allows for antibodies to be immobilized on the surface for specific detection, while the microfluidics system enables detection in a continuous flow with a negligible compromise between sensitivity and flow control over stabilization and mixing. Cytochrome c detection is used to illustrate the merits of the system. Jurkat cells are treated with the toxin staurosporine to trigger cell apoptosis and cytochrome c released into the cell culture medium is monitored via the newly invented optical microfluidic platform.

Label-free optical sensor systems have emerged that exhibit extraordinary sensitivity for detecting physical, chemical, and biological entities at the micro/nanoscale. Particularly exciting is the detection and analysis of molecules, on miniature optical devices that have many possible applications in health, environment, and security. These micro- and nanosensors have now reached a sensitivity level that allows for the detection and analysis of even single molecules. Their small size enables an exceedingly high sensitivity, and the application of quantum optical measurement techniques can allow the classical limits of detection to be approached or surpassed. The new class of label-free micro- and nanosensors allows dynamic processes at the single-molecule level to be observed directly with light. By virtue of their small interaction length, these micro- and nanosensors probe light-matter interactions over a dynamic range often inaccessible by other optical techniques. For researchers entering this rapidly advancing field of single-molecule micro- and nanosensors, there is an urgent need for a timely review that covers the most recent developments and that identifies the most exciting opportunities. The focus here is to provide a summary of the recent techniques that have either demonstrated label-free single-molecule detection or claim single-molecule sensitivity.

Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.

© 2017 Elsevier B.V.In this work a refractometric air pressure sensing platform based on spherical whispering-gallery mode microresonators is presented and analyzed. The sensitivity of this sensing approach is characterized by measuring the whispering-gallery mode spectral shifts caused by a change of air refractive index produced by dynamic sinusoidal pressure variations that lie between extremes of ±1.8kPa. A theoretical frame of work is developed to characterize the refractometric air pressure sensing platform by using the Ciddor equation for the refractive index of air, and a comparison is made against experimental results for the purpose of performance evaluation.

Whispering gallery mode biosensors have been widely exploited over the past decade to study molecular interactions by virtue of their high sensitivity and applicability in real-time kinetic analysis without the requirement to label. There have been immense research efforts made for advancing the instrumentation as well as the design of detection assays, with the common goal of progressing towards real-world sensing applications. We therefore review a set of recent developments made in this field and discuss the requirements that whispering gallery mode label-free sensors need to fulfill for making a real world impact outside of the laboratory. These requirements are directly related to the challenges that these sensors face, and the methods proposed to overcome them are discussed. Moving forward, we provide the future prospects and the potential impact of this technology.

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

AbstractPhotonic crystal modes can be tailored for increasing light matter interactions and light extraction efficiencies. These PhC properties have been explored for improving the device performance of LEDs, solar cells and precision biosensors. Tuning the extended band structure of 2D PhC provides a means for increasing light extraction throughout a planar device. This requires careful design and fabrication of PhC with a desirable mode structure overlapping with the spectral region of emission. We show a method for predicting and maximizing light extraction from 2D photonic crystal slabs, exemplified by maximizing silicon photoluminescence (PL). Systematically varying the lattice constant and filling factor, we predict the increases in PL intensity from band structure calculations and confirm predictions in micro-PL experiments. With the near optimal design parameters of PhC, we demonstrate more than 500-fold increase in PL intensity, measured near band edge of silicon at room temperature, an enhancement by an order of magnitude more than what has been reported.

AbstractSpectroscopy of whispering-gallery mode microresonators has become a powerful scientific tool, enabling the detection of single viruses, nanoparticles and even single molecules. Yet the demonstrated timescale of these schemes has been limited so far to milliseconds or more. Here we introduce a scheme that is orders of magnitude faster, capable of capturing complete spectral snapshots at nanosecond timescales—cavity ring-up spectroscopy. Based on sharply rising detuned probe pulses, cavity ring-up spectroscopy combines the sensitivity of heterodyne measurements with the highest-possible, transform-limited acquisition rate. As a demonstration, we capture spectra of microtoroid resonators at time intervals as short as 16 ns, directly monitoring submicrosecond dynamics of their optomechanical vibrations, thermorefractive response and Kerr nonlinearity. Cavity ring-up spectroscopy holds promise for the study of fast biological processes such as enzyme kinetics, protein folding and light harvesting, with applications in other fields such as cavity quantum electrodynamics and pulsed optomechanics.

In this Letter we propose the use of whispering gallery mode resonance tracking as a label-free optical means to monitor diffusion kinetics in glassy polymer microspheres. Approximate solutions to the governing diffusion equations are derived for the case of slow relaxation and small Stefan number. Transduction of physical changes in the polymer, including formation of a rubbery layer, swelling, and dissolution, into detectable resonance shifts are described using a perturbative approach. Concrete examples of poly(methyl methacrylate) and polystyrene spheres in water are considered.

A label-free method is developed to ratiometrically determine the stoichiometry of oligonucleotides attached to the surface of gold nanoparticle (GNP) by whispering gallery mode biosensing. Utilizing this scheme, it is furthermore shown that the stoichiometric ratio of GNP attached oligonucleotide species can be controlled by varying the concentration ratio of thiolated oligonucleotides that are used to modify the GNP.

In this work, we theoretically and experimentally investigate the thermal response of whispering gallery mode microresonators operating in an aqueous glycerol medium. Thermal stabilisation of the resonance wavelength is realised by appropriate choice of the resonator radius and glycerol concentration, with a 60 fold reduction in thermal sensitivity demonstrated. Finally, we employ our stabilised system to determine the thermal dependence of the molecular polarisability of adsorbed bovine serum albumin molecules and the refractive index of dextran and poly(diallyldimethylammonium chloride) coatings.

We present an erratum to correct inadvertent typographical errors in our paper [Adv. Opt. Photon. 7, 168 (2015)] and to update Fig. 7 therein following a revised version from the original authors.

Nucleic acid detection with label‐free biosensors circumvents costly fluorophore functionalization steps associated with conventional assays by utilizing transducers of impressive ultimate detection limits. Despite this technological prowess, molecular recognition at a surface limits the biosensors' sensitivity, specificity, and reusability. It is therefore imperative to integrate novel molecular approaches with existing label‐free transducers to overcome those limitations. Here, we demonstrate this concept by integrating a DNA strand displacement circuit with a micron‐scale whispering gallery mode (WGM) microsphere biosensor. The integrated biosensor exhibits at least 25‐fold improved nucleic acid sensitivity, and sets a new record for label‐free microcavity biosensors by detecting 80 pM (32 fmol) of a 22nt oligomer; this improvement results from the catalytic behavior of the circuit. Furthermore, the integrated sensor exhibits extremely high specificity; single nucleotide variants yield 40‐ to 100‐fold lower signal. Finally, the same physical sensor was demonstrated to alternatingly detect 2 different nucleic acid sequences through 5 cycles of detection, showcasing both its reusability and its versatility.

Combining graphene with plasmonic nanostructures is currently being explored for high sensitivity biochemical detection based on the surface‐enhanced Raman scattering (SERS) effect. Here, a novel and tunable platform for understanding SERS based on graphene monolayers transferred on arrays of split ring resonators (SRRs) exhibiting resonances in the visible range is introduced. Raman enhancement factors per area of graphene of up to 75 are measured, demonstrating the strong plasmonic coupling between graphene and the metamaterial resonances. Apart from the incident laser light, both the photoluminescence signal emitted by the SRRs and the Raman scattered light from graphene contribute to the excitation of distinct resonances, resulting in different SERS. This new perspective allows control of SERS in the case of graphene on plasmonic metamaterials or nanostructures and potentially paves the way towards an advanced SERS substrate that could lead to the detection of single molecules attached to graphene in future biochemical sensing devices.

Graphene on split ring resonator arrays, as described on page 151 by G. Sarau et al. represents a novel and tunable platform for gaining a basic understanding of surface-enhanced Raman scattering (SERS) effects. The Raman enhancement of graphene proves the strong plasmonic coupling between graphene and the metamaterial resonances excited by incident, photoluminescence, and Raman light. This opens the way towards an advanced SERS substrate for high-sensitivity detection of molecules attached to graphene. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

We present designs of all-optical reversible gates, namely, Feynman, Toffoli, Peres, and Feynman double gates, with optically controlled microresonators. To demonstrate the applicability, a bacteriorhodopsin protein-coated silica microcavity in contact between two tapered single-mode fibers has been used as an all-optical switch. Low-power control signals (

Optical resonator based biosensors are emerging as one of the most sensitive microsystem biodetection technology that boasts all of the capabilities for a next-generation lab-on-chip device: label-free detection down to single molecules, operation in aqueous environment and costeffective integration on microchips together with other photonic, electronic and fluidic components. We give a scholarly introduction to the emerging field of optical resonator based biosensing, review current applications,and explain how optical resonators are coated with biomolecules to construct logic devices. © Indian Institute of Science.

AbstractOptical microcavities that confine light in high-Q resonance promise all of the capabilities required for a successful next-generation microsystem biodetection technology. Label-free detection down to single molecules as well as operation in aqueous environments can be integrated cost-effectively on microchips, together with other photonic components, as well as electronic ones. We provide a comprehensive review of the sensing mechanisms utilized in this emerging field, their physics, engineering and material science aspects, and their application to nanoparticle analysis and biomolecular detection. We survey the most recent developments such as the use of mode splitting for self-referenced measurements, plasmonic nanoantennas for signal enhancements, the use of optical force for nanoparticle manipulation as well as the design of active devices for ultra-sensitive detection. Furthermore, we provide an outlook on the exciting capabilities of functionalized high-Q microcavities in the life sciences.

AbstractMicrocavity and whispering gallery mode (WGM) biosensors derive their sensitivity from monitoring frequency shifts induced by protein binding at sites of highly confined field intensities, where field strengths can be further amplified by excitation of plasmon resonances in nanoparticle layers. Here, we propose a mechanism based on optical trapping of a protein at the site of plasmonic field enhancements for achieving ultra sensitive detection in only microliter‐scale sample volumes, and in real‐time. We demonstrate femto‐Molar sensitivity corresponding to a few 1000 s of macromolecules. Simulations based on Mie theory agree well with the optical trapping concept at plasmonic ‘hotspots’ locations. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

We demonstrate lasing in GaAs photonic crystal waveguides with InAs quantum dots as gain medium. Structural disorder is present due to fabrication imperfection and causes multiple scattering of light and localization of light. Lasing modes with varying spatial extend are observed at random locations along the guide. Lasing frequencies are determined by the local structure and occur within a narrow frequency band which coincides with the slow light regime of the waveguide mode. The three-dimensional numerical simulation reveals that the main loss channel for lasing modes located away from the waveguide end is out-of-plane scattering by structural disorder.

We demonstrated a biosensing approach which, for the first time, combines the high sensitivity of whispering gallery modes (WGMs) with a metallic nanoparticle-based assay. We provided a computational model based on generalized Mie theory to explain the higher sensitivity of protein detection. We quantitatively analyzed the binding of a model protein (i.e. Bovine Serum Albumin) to gold nanoparticles from high-Q WGM resonance frequency shifts, and fit the results to an adsorption isotherm, which agrees with the theoretical predictions of a two-component adsorption model.

AbstractOptical resonator biosensors are emerging as one of the most sensitive microsystem biodetection technology that does not require amplification or labeling of the analyte. This minireview provides a scholarly introduction to this research area and reviews current advances in molecular diagnostics and nanoparticle detection.

We present designs of all-optical computing circuits, namely, half-full
adder/subtractor, de-multiplexer, multiplexer, and an arithmetic unit, based on
bacteriorhodopsin (BR) protein coated microcavity switch in a tree
architecture. The basic all-optical switch consists of an input infrared (IR)
laser beam at 1310 nm in a single mode fiber (SMF-28) switched by a control
pulsed laser beam at 532 nm, which triggers the change in the resonance
condition on a silica bead coated with BR between two tapered fibers. We show
that fast switching of 50 us can be achieved by injecting a blue laser beam at
410 nm that helps in truncating the BR photocycle at the M intermediate state.
Realization of all-optical switch with BR coated microcavity switch has been
done experimentally. Based on this basic switch configuration, designs of
all-optical higher computing circuits have been presented. The design requires
2n-1 switches to realize n bit computation. The proposed designs require less
number of switches than terahertz optical asymmetric demultiplexer based
interferometer designs. The combined advantages of high Q factor, tunability,
compactness and low power control signals, with the flexibility of cascading
switches to form circuits, makes the designs promising for practical
applications. The design combines the exceptional sensitivities of BR and
microcavities for realizing low power circuits and networks. The designs are
general and can be implemented (i) in both fiber-optic and integrated optic
formats, (ii) with any other coated photosensitive material, or (iii) an
externally controlled microresonator switch.

We show all-optical switching of an input infrared laser beam at 1310 nm by controlling the photoinduced retinal isomerization to tune the resonances in a silica microsphere coated with three bacteriorhodopsin (BR) protein monolayers. The all-optical tunable resonant coupler re-routes the infrared beam between two tapered fibers in 50 μs using a low power (<200 μW) green (532 nm) and blue (405 nm) pump beams. The basic switching configuration has been used to design all-optical computing circuits, namely, half and full adder/subtractor, de-multiplexer, multiplexer, and an arithmetic unit. The design requires 2n−1 switches to realize n bit computation. The designs combine the exceptional sensitivities of BR and high-Q microcavities and the versatile tree architecture for realizing low power circuits and networks (approximately mW power budget). The combined advantages of high Q-factor, tunability, compactness, and low power control signals, with the flexibility of cascading switches to form circuits, and reversibility and reconfigurability to realize arithmetic and logic functions, makes the designs promising for practical applications. The designs are general and can be implemented (i) in both fiber-optic and integrated optic formats, (ii) with any other coated photosensitive material, or (iii) any externally controlled microresonator switch.

The experimental observation of enhanced photoluminescence from high-Q silicon-based random photonic crystal microcavities embedded with PbSe colloidal quantum dots is being reported. The emission is optically excited at room temperature by a continuous-wave Ti-sapphire laser and exhibits randomly distributed localized modes with a minimum spectral linewidth of 4nm at 1.5μm wavelength.

. We report the label-free, real-time optical detection of Influenza a virus particles. Binding of single virions is observed from discrete changes in the resonance frequency/wavelength of a whispering-gallery mode excited in a microspherical cavity. We find that the magnitude of the discrete wavelength-shift signal can be sufficiently enhanced by reducing the microsphere size. A reactive sensing mechanism with inverse dependence on mode volume is confirmed in experiments with virus-sized polystyrene nanoparticles. By comparing the electromagnetic theory for this reactive effect with experiments, the size and mass (≈5.2 × 10
. −16
. g) of a bound virion are determined directly from the optimal resonance shift.
.

We present direct observations of electromagnetic fields localized in disordered photonic crystal waveguides and report the modal volumes and quality factors of the confined modes. Geometrical perturbations distributed uniformly throughout the crystal lattice were introduced by changing orientations of the polygonal lattice elements. Cavities in the disordered waveguides were excited by resonant coupling through a chain of random open resonators. Localized optical resonances with sub-(λ∕n)3 modal volumes and quality factors of up to ∼150000 were observed.

Photoinduced molecular transitions in bacteriorhodpsin are used to reversibly configure a micron-scale photonic component in which the optical response is resonantly enhanced. The chromophore retinal undergoes photoinduced all-trans to 13-cis conformational change, which tunes resonances in a silica microsphere coated with three bacteriorhodopsin monolayers. The tunable, all-optical resonant coupler reroutes a near-infrared beam (λprobe≅1311nm) between two tapered optical fibers using a low-power (<200μW) green pump (λpump=532nm). The approach represents a bottom-up paradigm for fabrication of hybrid molecular-photonic architectures that employ self-assembled biomolecules for optical manipulation at small scales.

We present an optical biosensor with unprecedented sensitivity for detection of unlabeled molecules. Our device uses optical resonances in a dielectric microparticle (whispering gallery modes) as the physical transducing mechanism. The resonances are excited by evanescent coupling to an eroded optical fiber and detected as dips in the light intensity transmitted through the fiber at different wavelengths. Binding of proteins on the microparticle surface is measured from a shift in resonance wavelength. We demonstrate the sensitivity of our device by measuring adsorption of bovine serum albumin and we show its use as a biosensor by detecting streptavidin binding to biotin.

. The TATA-binding protein (TBP)-related factor TRF1, has been
described in
. Drosophila
. and a related protein, TRF2, has
been found in a variety of higher eukaryotes. We report that human
(h)TRF2 is encoded by two mRNAs with common protein coding but distinct
5′ nontranslated regions. One mRNA is expressed ubiquitously
(hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted
expression pattern and is extremely abundant in testis. In addition, we
show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but
not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2.
Neither recombinant human (rh)TRF2 nor the native flag⋅hTRF2-TFIIA
complex is able to replace TBP or TFIID in basal or activated
transcription from various RNA polymerase II promoters. Instead,
rhTRF2, but not the flag⋅hTRF2–TFIIA complex, moderately inhibits
basal or activated transcription in the presence of rhTBP or
flag⋅TFIID. This effect is either completely (TBP-mediated
transcription) or partially (TFIID-mediated transcription) counteracted
by addition of free TFIIA. Neither rhTRF2 nor flag⋅hTRF2–TFIIA
has any effect on the repression of TFIID-mediated transcription by
negative cofactor-2 (NC2) and neither substitutes for TBP in RNA
polymerase III-mediated transcription.
.